Introduction

Cadmium is one of the toxic heavy metals of great environmental concern which enters the agricultural soil mostly through anthropogenic activities such as mining, sewage effluents, pesticides, chemical fertilizer application and industrial waste disposal (Singh et al. 2003; Kidd et al. 2007; Adewole et al. 2010; Mazharia and Homaeeb 2012; Hadi et al. 2014). From soil and water, it can easily be absorbed and accumulated in plant tissues due to its high bioavailability in soil and consequently reaches the human bodies through food chain (Alkorta et al. 2004; Liu et al. 2009; Ambedkar and Muniyan 2013). Crops cultivated in polluted soil may accumulate cadmium (Cd) in different parts, i.e. roots, leaves and fruits. Consumption of Cd-polluted plants may develop a number of Cd-related chronic diseases such as cancer, oxidative stress, tissue necrosis and impairment of the kidney and liver (Ogawa et al. 2004; Liu et al. 2005; Simmons et al. 2005; Åkesson et al. 2006; John et al. 2008; Kafel et al. 2014;). Heavy metals are not biodegradable (by microorganisms and plants) and continuously accumulate in soil, and their presence in soil (especially agricultural soil) is of great concern for both plant and animal health (Mubeen et al. 2010; Okedeyi et al. 2014). For clean and sustainable environment, the removal of toxic heavy metals from soil and water is very important and needs the development of effective, affordable and environment-friendly technologies. Various conventional methods (including both chemical and physical) have been used for the restoration of heavy metal-contaminated soil, but these methods are very costly and laborious and adversely affect both the soil structure and ecosystem. The discovery of some plants having ability to accumulate and tolerate high concentrations of heavy metals led to the development of a new plant-based technology, known as phytoremediation (Reeves and Brooks 1983; Baker and Brooks 1989; Entry et al. 1999). Phytoremediation technology is cost effective, solar driven, aesthetically pleasing and environment friendly (Schwitzguebel et al. 2009; Chai et al. 2012). More than 400 species of plants have been investigated for their heavy metal phytoremediation potential, and most of these plants belong to Arabidopsis, Brassica, Sedum and Thlaspi species (Lone et al. 2008). In the present research, Parthenium hysterophorus was studied at its reproductive stage for its Cd phytoextraction potential. This plant belongs to Asteraceae family and is native species of America, which invaded Australia, India, Pakistan and some parts of Africa (Parsons and Cuthbertson 1992; Dhawan and Dhawan 1996). It is a fast-growing, stress-tolerant perennial herb, which is unpalatable to herbivores, thus prevents metal entrance into food chain. In previous literature, Parthenium has been used for lead (Pb) phytoextraction (Hadi and Bano 2009).

Plants grown on metal-contaminated soil often show slow growth and lower biomass and accumulate lower metal concentration within the biomass (Persans and Salt 2000; Li et al. 2003). For efficient heavy metal phytoremediation, plant should have high biomass and can also tolerate and accumulate high concentration of toxic heavy metal within their tissues. Heavy metal-tolerant plants generally have lower biomass or most plants showing high biomass do not show tolerance to heavy metals in soil. To increase biomass as well as metal phytoextraction potential of plants, several chemical modifications (applied to plant or added to soil/ water) have been done by several scientists, such as the application of hormones and metal chelators (Kamnev and Van Der Lelie 2000; Chen and Cutright 2001; Hadi and Bano 2009; Falkowska et al. 2011). In the present experiment, a plant hormone gibberelic acid (GA3) and a synthetic chelator ethylenediaminetetraacetic acid (EDTA) were used for increasing the Cd phytoremediation potential of the plant. GA3 enhances plant growth and biomass while EDTA increases the metal bioavailability in soil by forming complexes with metals (Benjerano and Lips 1970; Broughton and McComb 1971; Chen et al. 2004; Hadi and Bano 2009; Hadi et al. 2010).

Plant under stress conditions produce and accumulate a variety of metabolic products including amino acids (such as proline) and phenolic compounds (Grace and Logan 2000; Diaz et al. 2001; Sakihama and Yamasaki 2002). Many investigators have reported accumulation of free proline under conditions of salinity, drought, intense light and ultraviolet radiation and heavy metals and in response to oxidative stress and biotic stresses (Fabro et al. 2004; Choudhary et al. 2005; Haudecoeur et al. 2009; Yang et al. 2009). Proline not only takes part in protein synthesis but also showed a positive correlation with plant stress tolerance. Proline maintains osmotic or cell turgor pressure, reduces electrolyte leakage by stabilizing membranes and protects plant from oxidative stress by reducing concentration of reactive oxygen species (ROS) (Xu et al. 2009; Hayat et al. 2012). Similarly, phenolic compounds are produced during heavy metal stress and act as antioxidant and directly scavenge ROS (Michalak 2006). ROSs can destroy lipids, DNA, proteins and chlorophyll by producing highly reactive (nascent) oxygen (Ramadevi and Prasad 1998). High concentrations of phenolic compound have been reported in different plants such as wheat in response to nickel toxicity (Diaz et al. 2001), Phaseolus vulgaris when exposed to cadmium, Phyllanthus tenellus leaves in response to copper sulphate (Diaz et al. 2001) and maize due to aluminium (Winkel-shirley 2002).

The present study was carried out with objectives to evaluate the effect of plant growth regulator (gibberellic acid, GA3) and a chelating agent (EDTA) either alone or in different combinations (synergistic effect) on (1) growth and biomass of the P. hysterophorus plant in Cd-contaminated soil; (2) concentration of proline and phenolics in different parts of the plant; (3) Cd absorption, its translocation into plant shoot and accumulation in different parts of the plant; (4) effect of Cd on contents of chlorophyll in leaves; and (5) correlation of total phenolics and free proline with dry biomass and Cd contents of plant parts.

Materials and methods

Preparation of soil and addition of cadmium

Soil was collected from fields nearby the University of Malakand at Chakdara, Pakistan. The soil was grounded into powdered form after drying in sunlight. Water holding capacity (300 ml water/kg soil ± 3) and pH (6.5 ± 0.3) of the soil was calculated. The dried soil was then added into plastic pots (18 cm height × 15 cm diameter) at the rate of 1 kg soil/ pot. Cadmium (100 mg/kg soil) was added to each pot as cadmium acetate dihydrate (CH3COO)2Cd·2H2O (Merck, Germany) solution and allowed for 2 months. No cadmium was added to the control (C) pots.

Transplantation of seedlings and plant growth

Each pot was watered a day before transplantation of plantlets. Seedlings (P. hysterophorus plantlets) of uniform size were selected and single plantlet was transferred into each pot. Three replicate pots were used for each treatment and controls. Two controls were used, one without cadmium (C) and the other with cadmium only (C1). C and C1 were compared for the effect of Cd on growth. Plants were watered, at 3-day interval. Plants were grown under natural condition of light and temperature (35/25 °C).

Treatments used

The plants were treated as given in Table 1.

Table 1 Treatments done during the whole experiment
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Exogenous application of GA3

Three different concentrations (10−2, 10−4 and 10−6 M) of GA3 were applied to the plants in the form of foliar spray (10 ml/plant) in four doses (each dose at 10-day intervals). First treatment was made 10 days after transplantation. Polythene bags were used to cover soil in pots during application of GA3 so that hormone droplets will not reach into the root zone.

EDTA addition into soil

A total of 40 mg of EDTA was added per kilogramme soil (i.e. single pot) in the form of aqueous solution in two different ways, i.e. single dose of 40 mg EDTA/pot (kg soil) and in four split doses, each of 10 mg EDTA/dose, at 10-day interval. First treatment of EDTA (single or split dose) was made 10 days after transplantation.

Combination treatments of GA3 and EDTA

Some plants were treated with both GA3 and EDTA in combination. Three different concentrations of GA3 (10−6, 10−4 and 10−2 M) and two different ways of EDTA (40 mg/pot) application (single and split doses) were used in six types of different combinations (3 × 2). In the combination treatments, both the GA3 and EDTA applications were made as mentioned earlier.

Plant growth parameter analysis

Plant shoot length was measured on weekly basis. After two and a half months from transplantation (at flowering stage), the plants were harvested and lengths of the plants root and shoot were measured using a centimetre ruler. Plants were washed with a 5 mM solution of EDTA and Tris-HCl (pH 6.0) and then rinsed with distilled water to remove any surface-bounded metal (Genrich et al. 2000). After washing, each plant was cut into three parts, i.e. roots, stem and leaves, and fresh biomass of each part was measured with the help of analytical balance. The parts of each plant were packed in separate paper envelopes and then dried in oven for 48 h at 80 °C. The dry biomass of each part was measured by analytical balance and then grinded into powdered form.

Analysis of free proline in plant root and leaves

Bates et al. (1973) method was used for the quantification of free proline within different parts (root and leaves) of three replicate plants. Fresh plant tissue (100 mg from each part) was homogenized/crushed in 2-ml tubes containing 1.5 ml of 3 % sulfosalicylic acid. The homogenate was then centrifuged for 5 min at 13,000 rpm. The supernatant (only 300 μl) was transferred into new tube and then 2 ml each of acid ninhydrin (containing 1.25 g of ninhydrin heated in 20 ml of phosphoric acid (6 M) and 30 ml of glacial acetic acid until dissolved completely) and glacial acetic acid were added to it. The mixture was kept in water bath (100 °C) for 1 h. The tubes were immediately dipped into ice after removing from water bath. Toluene (1 ml per tube) was added to the reaction mixture and then vigorously mixed for 10–30 s. Toluene containing chromophore layer was removed from the aqueous phase with the help of micropipette and warmed to the room temperature. Spectrophotometer was used (250-nm wavelength) to measure the absorbance of each sample. Toluene was used as a blank (control). The standard curve was used to calculate the concentration of proline in samples. Three replicates were used for each sample.

Total phenolic estimation in roots and leaves

Total phenolics were calculated in root, stem and leaves of three replicate plants. Dried sample (200 mg each) was mixed with 10 ml of methanol (80 %) and then shake for at least 30 min in close vessel (flask) to prevent evaporation of solvent. From each extract, 2 ml was taken in separate tubes and centrifuged at 13,000 rpm for 3 to 5 min. Singleton and Rossi (1965) method with slight modifications was used for analysis of total phenolics in extract. Folin-Ciocalteau (FC) reagent (250 μl) was mixed with 100 μl gallic acid standard solutions or methanolic extract, and the mixture was kept in dark (at room temperature) for 3–5 min. Then 7 % (500 μl) sodium carbonate (Na2CO3) solution was added to the mixture, and dH2O was used and raised the net volume up to 5 ml. The mixture was kept in dark at room temperature for 2 h. Spectrophotometer was used to measure the absorbance of the samples at 760 nm. Different standard solutions (0, 10, 30, 50, 100, 150 mg/l) of gallic acid were prepared in methanol (80 %), and their absorbances were used as standard for measuring total phenolics in each sample. Methanol solution with a concentration of 80 % was used as blank (control). Three replicates were used for each sample.

Chlorophyll estimation in leaves

Concentration of chlorophyll a and b was measured by using the method of Arnon (1949). First of all, fresh leaves were obtained from the plants (both control and treated plants). Then 2 ml of acetone (80 %) was mixed with 200 mg of fresh leaves and properly grinded. After grinding, the mixtures were shifted into Ependorf tubes and then centrifuged for 5 min at 10,000 rpm. The supernatant (after centrifugation) was poured into clean test tubes and 6 ml of acetone (80 %) was added to it. The samples were then analysed for absorbance at 645 and 663 nm in spectrophotometer. The following formulas were used for calculating the concentration of chlorophyll a and b:

$$ \mathrm{Chlorophyll}\;\underset{\bar{\mkern6mu}}{\mathrm{a}}\left(\upmu \mathrm{g}/\mathrm{ml}\right)=12.7\left({A}_{663}\right)-2.69\left({A}_{645}\right) $$
$$ \mathrm{Chlorophyll}\;\underset{\bar{\mkern6mu}}{\mathrm{b}}\left(\upmu \mathrm{g}/\mathrm{ml}\right)=22.9\left({A}_{645}\right)-4.68\left({A}_{663}\right) $$

Cd analysis in different plant parts

Oven-dried samples (root, stem and leaves) were first grounded into powdered form and then subjected to acid digestion using Allen’s (1974) method. Dried powder (0.25 g) from each sample was taken into separate flasks (50 ml). Three-acid mixture (6.5 ml) containing sulfuric acid, nitric acid and perchloric acid (1, 5 and 0.5 ml, respectively) was added to each flask. For complete digestion, each flask (sample) was kept on electric hot plates until completely digested. The digested samples were then filtered into another volumetric flask (50 ml), and with the help of dH2O, the volume was raised up to 50 ml. Each filtrate sample was then stored in small plastic bottles. The samples were then analysed for Cd concentration with the help of atomic absorption/flame spectrophotometer (model Hitachi Z-8000, Japan). Three replicate plants per treatment were analysed for Cd content.

Statistical analysis

The data was subjected to analysis of variance (ANOVA) and correlations between parameters. Tukey’s HSD test (p ≤ 0.05) was used for checking significant differences between means. SPSS 16 and MS Excel 2007 were used.

Results

Effect of GA3 and EDTA treatments on length (root and shoot), biomass (fresh and dry) and water contents of P. hysterophorus plant under Cd stress

Plant length, biomass and water content were significantly reduced by Cd addition into soil (100 mg Cd/kg) when control C (without Cd) and C1 (with Cd only) were compared, except the dry biomass (DBM) of stem and total water content (TWC) of leaves (Table 2). The effect of all treatments (except EDTA-treated plants T4, T5) significantly increased the root and shoot length on Cd-contaminated soil when compared to C1 (Cd only) (Fig. 1). The highest significant root length (24.33 ± 1.00 cm) and stem length (44.33 ± 4.73 cm) was demonstrated by the treatment T3 (GA3 10−6 M, foliar spray). Fresh biomass (FBM) and TWC of root significantly increased in all treatments (except T10 and T11), while FBM of stem and leaves were significantly higher only in GA3-alone treatments (T1, T2 and T3) as compared to C1 (Table 2). All treatments significantly increased DBM of root, stem, leaves and entire plant, and the highest significant DBM in roots (1.65 ± 0.02 g), stem (2.40 ± 0.05 g), leaves (2.40 ± 0.05 g) and entire plant (6.45 ± 0.12 g) was recorded in treatment T3 (Cd + GA3 10−6 M) as given in Table 2.

Table 2 Effect of different treatments of GA3 and EDTA on different growth parameters (length, biomass and water content) of Parthenium hysterophorus plant in cadmium-contaminated soil (100 mg Cd/kg soil)
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Fig. 1
figure 1

Effect of different treatments of GA3 and EDTA on root and shoot length of Parthenium hysterophorus plant, in cadmium-contaminated soil (100 mg Cd/kg soil). C (without Cd), C1 (with Cd), T1 (Cd + GA3 10−2 M), T2 (Cd + GA3 10−4 M), T3 (Cd + GA3 10−6 M), T4 (Cd + EDTA 40 mg), T5 (Cd + EDTA 10 mg), T6 (Cd + GA3 10−6 M + EDTA 40 mg), T7 (Cd + GA3 10−6 M + EDTA 10 mg), T8 (Cd + GA3 10−4 M + EDTA 40 mg), T9 (Cd + GA3 10−4 M + EDTA 10 mg), T10 (Cd + GA3 10−2 M + EDTA 40 mg), T11 (Cd + GA3 10−2 M + EDTA 10 mg)

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Effect of different treatments of GA3 and EDTA on Cd phytoaccumulation

Root cadmium concentration of the plant was increased significantly in all the treatments when compared with C1 (control with Cd only), and the highest significant root Cd concentration (1267.00 ± 12.60 and 1245 ± 16.20 ppm) was recorded for the EDTA treatments T4 and T5, respectively (Table 3). The treatment T11 (Cd + GA3 10−2 M + EDTA split doses) significantly increased the highest Cd concentration in stem and leaf (166.33 ± 18.00 and 570.00 ± 23.45 ppm, respectively). Accumulation of Cd (mg/DBM) in different parts of the plant was high significantly in all treatments (except in T1 plant stem) as compared to C1 (Table 3). The high significant Cd accumulation in roots was found in GA3 and EDTA combination treatments T9, T10 and T11, respectively, 0.84 ± 0.04, 0.86 ± 0.03 and 0.87 ± 0.04 mg Cd/DBM. The treatment T11 showed the highest Cd accumulation in stem (0.26 ± 0.03 mg Cd/DBM), leaves (0.83 ± 0.04 mg Cd/DBM) and entire plant (1.97 ± 0.11 mg Cd/DBM). The treatments showed an increase of 4.07–9.79-fold Cd contents in roots, 1.61–3.21 in stem, 1.86–4.71 in leaves and 2.37–5.65-fold in entire plant Cd accumulation compared to C1 as given in Table 3. The highest increase in Cd accumulation within roots (9.79 times), stem (3.21 times), leaves (4.71 times) and entire plant (5.65 times) was demonstrated by the treatment T11 (Cd + GA3 10−2 M + EDTA split doses). The results showed that the highest Cd accumulation percentage was found within roots followed by leaves of the plant while the lowest Cd accumulation percentage was noted in the plant stem (Table 3). Cadmium bioconcentration factor (BCF) of the plant being higher than the one (i.e. 1.85 ± 0.22) in the control C1 plants shows that P. hysterophorus is a hyperaccumulator of Cd. The treatment further increased the Cd BCF and the increase was found statistically significant as compared to the control C1. The highest significant Cd BCF (9.75 ± 0.34) was demonstrated by the treatment T11 (Cd + GA3 10−2 M + EDTA split doses).

Table 3 Effect of different treatments of GA3 and EDTA on Cd concentration, accumulation, translocation and bioconcentration of Parthenium hysterophorus plant in cadmium-contaminated soil (100 mg Cd/kg soil)
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Effect of exogenous GA3 and EDTA on total phenolics, free proline and chlorophyll (a/b) under Cd stress

Free proline and total phenolics in roots, stem and leaves while chlorophyll (a/b) contents in leaves of the plant are presented in Table 4. Cadmium added to the soil significantly increased free proline contents in plant roots while total phenolics were significantly higher in all parts (root, stem and leaves) of the plant (comparing C with C1). Free proline in stem increased significantly in T3 (GA3 treatment) and in leaves of combination treatments (T10 and T11) when compared with control C1 (Table 4). The total phenolics in roots significantly increased with GA3 treatments while EDTA treatments (T4 and T5) showed non-significant increase in phenolics (compared to control C1) as given in Table 4. In leaves, all the treatments showed significant increase in total phenolics (compared to C1). The highest significant total phenolic content in roots (79.00 ± 3.94 ppm) was found in treatment T7 while in stem (34.00 ± 2.50 ppm) and leaves (156.00 ± 13.20 ppm), it was recorded in the treatment T11 (Table 4). The chlorophyll (a/b) was reduced significantly in plant on cadmium-polluted soil (comparing C with C1). The treatments containing GA3 foliar spray significantly increased chlorophyll (a/b) concentration in leaves of the plant as compared to C1, except the chlorophyll a contents of T9, T10 and T11 (where the increase is non-significant compared to C1). EDTA-alone treatments showed significant decrease in chlorophyll content as compared to C1 (Table 4).

Table 4 Effect of gibberellic acid and EDTA treatments on free proline, total phenolics and chlorophyll (a/b) contents of Parthenium hysterophorus plant grown in Cd-contaminated soil (100 mg Cd/kg soil)
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Correlations among different parameters measured

Correlations among different parameters measured in roots of the plant are presented in Table 5. The table shows significantly positive correlations among certain parameters (length, FBM, DBM and TWC). The correlation of root Cd concentration with length (R 2 = −0.452), FBM (R 2 = −0.588), DBM (R 2 = −0.674) and TWC (R 2 = −0.546) of the plant roots was found negative. Cadmium concentration in roots showed highly significant positive correlation (R 2 = 0.661) with the accumulation of Cd in roots. The concentration of free proline and total phenolics in roots showed a positive significant correlations (R 2 = 0.527 and R 2 = 0.554, respectively) with Cd accumulation, but their correlation with the concentration of Cd in roots was non-significant. Total phenolics of roots showed positive significant correlations with length (R 2 = 0.728), FBM (R 2 = 0.537) and TWC (R 2 = 0.590) while its correlation with roots DBM was positive but non-significant. Proline contents of roots demonstrated positive correlation with the growth parameters but were non-significant. Table 6 shows positive and significant correlations among length, FBM, DBM and TWC of the stem, while the correlations of these parameters with stem Cd concentration were negative. Stem Cd accumulation showed negative correlations with growth parameters. Free proline and total phenolic concentration of stem showed positive correlation with all the parameters measured in plant stem, but the correlations of proline concentration was statistically significant only with phenolic content (R 2 = 0.577) and Cd accumulation (R 2 = 0.867). The correlations of total phenolics were significant with all the parameters except Cd concentration and accumulation in stem. Table 7 demonstrates correlations among different parameters measured in plant leaves. Like roots and stem, the leaves of plant showed positive significant correlations among the growth parameters (FBM, DBM and TWC). Cadmium concentration and accumulation of leaf demonstrated negative correlations with the growth parameters. Correlation between free proline and total phenolics of leaves was positive and significant. The correlations of chlorophyll with growth parameters were positive and were negative with Cd concentration.

Table 5 Correlations among different parameters measured in roots of P. hysterophorus plant
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Table 6 Correlations among different parameters measured in stem of P. hysterophorus plant
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Table 7 Correlation among different parameters measured in leaves of P. hysterophorus plant
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Discussion

Plant growth and biomass

Plant growth and biomass have been reported to be highly sensitive to heavy metal stress (Arun et al. 2005; Hadi and Bano 2009; John et al. 2009; Hadi et al. 2010). Heavy metal like cadmium in plant tissues reduces plant growth due to toxicity (Khatamipour et al. 2011). In the present investigation, cadmium demonstrated significant reduction in growth and biomass of P. hysterophorus plant (comparing C with C1). Decrease in growth of plants in cadmium-contaminated soil is often observed, and this reduction in length (of root and shoot) and biomass could be directly related to the negative effect of heavy metal on the division of meristematic cells and on the cell elongation and expansion (Houshmandfar and Moraghebi 2011). One of the reasons for inhibition of cell elongation might be the effect of metals on cell wall components and their structures (Poschenrieder et al. 1989). Present results showed reduction in fresh biomass and consequently in the water contents within different plant parts under cadmium stress (comparing C with C1). Similar reduction in fresh biomass under cadmium stress was reported by Zheng et al. (2010) and Khatamipour et al. (2011) in Glycyrrhiza uralensis plant. Cadmium has also been found to cause physiological drought by altering water content in plant tissues (Barcelo and Poschenriede 1990).

Addition of EDTA into the cadmium-contaminated soil further reduced the plant growth while application of GA3 foliar spray enhanced the growth and biomass of the P. hysterophorus plant. This decreasing effect of EDTA on plant growth and biomass might probably be due to increase in mobility of cadmium by EDTA in soil (Lou et al. 2007; Epelde et al. 2008). The increase in growth and biomass might be due to the role of GA3 in promotion of cell enlargement (Buchanan et al. 2000) and on the rate of cell division (Moore 1989; Arteca 1996), two main processes for the increase in growth and biomass. GA3 enhances the synthesis of DNA, RNA and protein (Benjerano and Lips 1970; Broughton and McComb 1971) and ribose and polyribosome multiplication (Evins and Varner 1972) would increase biomass of a plant. GA3 treatment also increases permeability of cell membrane (Wood and Paleg 1974; Crozier and Turnbull 1984) that would enhance absorption of mineral nutrients and their transport and utilization (Crozier and Turnbull 1984; Aloni et al. 1986; Al-Wakeel et al. 1995; Ansari 1996; Khan et al. 1998), thus, enhancing the capability of the GA3-treated plants for high biomass production as demonstrated in our experiment (Table 2). Increase in biomass due to GA3 application has been observed in tomato (Masroor et al. 2006) and in maize plant (Hadi et al. 2010).

Plant cadmium contents

The results showed that P. hysterophorus plant is a hyperaccumulator of cadmium at its flowering stage obvious from its high bioconcentration factor (1.85). GA3 and EDTA in combination greatly increase cadmium concentration within different parts of the plant. The reason might be that EDTA increases the metal bioavailability in soil solution (Elless and Blaylock 2000; Chen and Cutright 2001; Thayalakumaran et al. 2003; Meers et al. 2005; Mamindy-Pajany et al. 2014;) while the GA3 increases absorption and translocation of cadmium into different parts of the plant (Tassi et al. 2008; Hadi et al. 2014).

Proline concentration

Accumulation of proline in plant tissue is often considered as an indicator of environmental stress such as drought, salinity and heavy metal stress. It has been found that free proline chelates cadmium ion in plant tissues and converts them into non-toxic complex of Cd-proline (Sharma et al. 1998). Our results showed a strong correlation between free proline and cadmium accumulation within different tissues of the P. hysterophorus plant. This suggests that free proline may play an important role in Cd accumulation and also in the reduction of cadmium toxicity within plants. Several plants have been reported to accumulate high concentration of free proline under heavy metal stress such as sunflower, wheat, tomato, milk thistle, Solanum nigrum and Vigna unguiculata (Lalk and Dorfling 1985; Bhattacharjee and Mukherjee 1994; Costa and Morel 1994; De and Mukherjee 1998; Zengin and Munzuroglu 2006; Sun et al. 2007; Khatamipour et al. 2011). Higher concentration of free proline was recorded in the roots of P. hysterophorus plant as compared to the stem and leaves. Similarly high root proline concentration in V. unguiculata plant was found by Bhattacharjee and Mukherjee (1994). GA3 treatment demonstrated high concentrations of free proline, which suggest an important role of GA3 in the synthesis of proline.

Phenolic concentration within plant tissues

Phenolic compounds play an important role in protection, restoration and degradation processes caused by toxic chemicals (Rice-Evans et al. 1997). High concentration of total phenolics has been found in different plants under various environmental stresses (Grace and Logan 2000; Lavola et al. 2000; Diaz et al. 2001; Sakihama and Yamasaki 2002). Soluble phenolic compounds showed important antioxidant activity and are thus considered to be closely related to stress situations (Wild and Schmitt 1995). Schwitzguébel et al. (2001) reported that Scots pine accumulates high concentration of soluble phenolics subjected to Cd stress. Phenolic compounds act as antioxidant during heavy metal stress (Michalak 2006). High concentration of phenolics was recorded in leaves of the P. hysterophorus plant compared to roots and stem. Similarly high concentration of phenolic compounds in leaves of Crotalaria juncea was reported (Uraguchi et al. 2006).

Chlorophyll contents

Excess of cadmium in soil decreases content of chlorophyll (Ngayila et al. 2008), its synthesis rate (Vajpayee et al. 2000), efficiency of photosystems (Chugh et al. 1997), photosynthetic enzymes (Mobin and Nafees 2007; Thapar et al. 2008) and plant water balance and consequently reduces plant growth and biomass (Zhou and Qiu 2005). Present results demonstrated that negative correlation existed between cadmium contents of leaf and the chlorophyll contents (Table 7) which are in agreement with earlier reports of Mobin and Nafees (2007), Sun et al. (2008), Ekmekci et al. (2008) and Xue et al. (2014), who found that heavy metal suppressed the photosynthetic activity of plants. Faller et al. (2005) demonstrated that Cd2+ has inhibitory effect on the photoactivation of photosystem II as result of its competitive binding with the Ca2+ site. The net photosynthetic rate has been shown to decrease conspicuously with high concentrations of cadmium (Lakshaman and Surinder 1999). Different physiological activities influence the metabolization of chlorophyll in plants. The chlorophyll a was first synthesized and transformed into chlorophyll b (Guo et al. 2006). Present results showed higher content of chlorophyll a in Parthenium plant as compared to the chlorophyll b (Table 4) and showed agreement with the findings of Mobin and Nafees (2007).

Conclusions and recommendations

Phytoextraction capabilities of P. hysterophorus plant highly increased at flowering stage (reproductive stage), and on bases of its bioconcentration factor, it is suggested as hyperaccumulator of cadmium. GA3 increased cadmium accumulation in plant. The effect of GA3 was more pronounced at higher concentration in combination with split doses of EDTA at low concentrations. Free proline and total phenolics significantly increased with the increase in Cd concentration in plant tissues, especially in the GA3-treated plants. This suggests that GA3 has some role in the synthesis of these compounds. Proline and phenolics showed positive correlation with the plant dry biomass as well as with the Cd accumulation in different parts of the plant. Further study is recommended to investigate the biochemical and molecular basis of proline and phenolic synthesis and the mechanism through which GA3 enhance their biosynthesis in plant during Cd stress.